Brain Imaging and Behavior (2018) 12:1795–1803

https://doi.org/10.1007/s11682-018-9850-z

ORIGINAL RESEARCH

Difference in regional neural fluctuations and functional connectivity

in Crohn’s disease: a resting-state functional MRI study
Chunhui Bao1 · Peng Liu2 · Huirong Liu3 · Xiaoming Jin4 · Yin Shi3 · Luyi Wu1 · Xiaoqing Zeng5 · Jianye Zhang6 ·
Di Wang1 · Vince D. Calhoun7,8 · Jie Tian2 · Huangan Wu1

Published online: 20 February 2018

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Patients with Crohn’s disease (CD) are shown to have abnormal changes in brain structures. This study aimed to further
investigate whether these patients have abnormal brain activities and network connectivity. Sixty patients with CD and 40
healthy controls (HCs) underwent resting-state functional magnetic resonance imaging (fMRI) scans. Amplitude of low-
frequency fluctuation (ALFF) and seed-based functional connectivity (FC) were used to assess differences in spontaneous
regional brain activity and functional connectivity. Compared to the HCs, patients with CD showed significantly higher
ALFF values in hippocampus and parahippocampus (HIPP/paraHIPP), anterior cingulate cortex, insula, superior frontal
cortex and precuneus. The ALFF values were significantly lower in secondary somatosensory cortex (S2), precentral gyrus,
and medial prefrontal cortex. Functional connectivities between left HIPP and left inferior temporal cortex, and right mid-
dle cingulate cortex, HIPP, and fusiform area were significantly lower. The functional connectivities between right HIPP
and right inferior orbitofrontal cortex and left HIPP were also significantly lower. Patients with CD showed higher or lower
spontaneous activity in multiple brain regions. Altered activities in these brain regions may collectively reflect abnormal
function and regulation of visceral pain and sensation, external environmental monitoring, and cognitive processing in these
patients. Lower functional connectivity of the hippocampus-limbic system was observed in these patients. These findings
may provide more information to elucidate the neurobiological mechanisms of the disease.

Keywords Crohn’s disease · Resting-state functional MRI · Amplitude of low-frequency fluctuation · Functional
connectivity

Chunhui Bao and Peng Liu contributed equally to this work.

4
* Huirong Liu Stark Neurosciences Research Institute, Indiana University
lhr_tcm@139.com School of Medicine, Indianapolis, IN 46202, USA
5
* Huangan Wu Department of Gastroenterology, Zhongshan Hospital, Fudan
wuhuangan@139.com University, Shanghai 200032, China
6
1 Department of Radiology, Shanghai Mental Health
Key Laboratory of Acupuncture and Immunological Effects,
Center, Shanghai Jiaotong University School of Medicine,
Shanghai University of Traditional Chinese Medicine,
Shanghai 200030, China
Shanghai 200030, China
7
2 The Mind Research Network, Albuquerque, NM 87131, USA
Life Sciences Research Center, School of Life Sciences
8
and Technology, Xidian University, Xi’an, Shaanxi 710071, Department of Electrical and Computer Engineering,
China University of New Mexico, Albuquerque, NM 87131, USA
3
Outpatient Department, Shanghai Research Institute
of Acupuncture and Meridian, Shanghai University
of Traditional Chinese Medicine, Shanghai 200030, China

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Introduction
Crohn’s disease (CD), a type of inflammatory bowel dis-
ease (IBD), is a chronic gastrointestinal inflammatory dis-
ease of unknown etiology. The major signs and symptoms
include abdominal pain, chronic diarrhea, weight loss and
fatigue. The disease typically has progressive course and
causes intestinal damage or even disability in severe cases
(Torres et al. 2016). Currently, there is no definitive treat-
ment for CD. The condition typically requires lifelong
medication and supportive care. Recent years have wit-
nessed a steady increase in the incidence of IBD in Asia;
China has the highest incidence of IBD in Asia, which
places a heavy burden on health care resources and affects
social productivity (Ng et al. 2013). Because chronic pain,
inflammation, and brain-gut interaction are known to
involve various brain networks (Vermeulen et al. 2014),
characterizing specific changes in brain network and
activity in CD patients is important for understanding the
mechanism of the disease and controlling the symptoms.
Functional abnormality in the central nervous system
and the brain-gut axis was recently shown to play a key
role in the pathogenesis and development of CD (Al and
Aziz 2014; Bonaz and Bernstein 2013). Studies have
shown abnormal brain activity in CD patients in remis-
sion. Specifically, altered resting-state brain activity was
observed in brain regions involved in visceral sensation,
motility, emotional awareness, attention and cognition
(Bao et al. 2016a, b). However, different brain activities
may be related to different symptoms and pathogenesis
of CD. A previous study showed that abnormal brain
activity may be related to abdominal pain sensation in
CD patients. Among patients with CD in remission, those
with and without abdominal pain showed differences in
resting-state brain activity; further, activity profile in spe-
cific brain areas were related to the severity of the disease
(Bao et al. 2016a, b). Researchers have also investigated
functional brain activity evoked by psychological stress
in patients with CD in remission. They found altered neu-
ral activity in hippocampus, amygdala, and insula in CD
patients (Agostini et al. 2013). However, functional brain
activity in patients with CD is not well characterized. The
key regions with abnormal brain activity in these patients
have not been fully elucidated and findings from previous
studies need to be further validated.
Resting-state functional magnetic resonance imaging
(rs-fMRI) has emerged as a useful technique to study
the changes in functional activities in the resting state of
brain. Amplitude of low frequency fluctuations (ALFF),
which detect the amplitude of blood oxygen level depend-
ent (BOLD) signal relative to the baseline (Zang et al.
2007), can be used to reflects the level of spontaneous
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Brain Imaging and Behavior (2018) 12:1795–1803
activity at each voxel. Functional connectivity (FC), which
reflects synchronous spontaneous fluctuations between
brain regions, can provide information about functional
integrity of brain networks (Fox and Raichle 2007). The
combination of these two methods allows assessment of
the intrinsic activity of a specific brain region and its net-
work associated with a disease. Having been widely used
in the study of functional gastrointestinal disorders (Ma
et al. 2015; Zhou et al. 2013), neurological disorders (Li
et al. 2016; Van Hees et al. 2014), psychosis (Li et al.
2014; Zhang et al. 2014), and other medical conditions,
this method shows promise in investigating functional
activity of the resting brain. To date, no study has been
conducted on resting-state brain activity indexed by ALFF
and FC in patients with CD. Therefore, these approaches
may help elucidate the characteristics of resting-state brain
activity in CD patients, and provide useful information on
the pathophysiological mechanisms of CD.
In this study, we used ALFF and FC to explore resting-
state brain activity in patients with CD in remission. We
hypothesized that there would be differences in ALFF and
FC in certain brain regions between patients with CD in
remission and healthy controls, and that neuroimaging
results would be associated with the duration of disease.
Methods
Subjects
This study was approved by the Ethics Committee of the
Yueyang Hospital of Integrated Traditional Chinese and
Western Medicine affiliated with Shanghai University of
Traditional Chinese Medicine. All subjects signed informed
consent forms.
Sixty patients with CD were recruited from the outpa-
tient clinics for inflammatory bowel diseases at the Shang-
hai Research Institute of Acupuncture and Meridian, and
the Endoscopy Center of Zhongshan Hospital affiliated
with Fudan University. All subjects underwent systematic
and gastrointestinal screening, including colonoscopy and
intestinal mucosal biopsy. CD Endoscopic Index of Severity
(CDEIS) of each subject was scored by an expert gastroen-
terologist. To assess the level of inflammation, C-reactive
protein, erythrocyte sedimentation rate and platelet counts
for each subject were measured and recorded two weeks
prior to fMRI scans.
Inclusion criteria: right-handed (as determined by Edin-
burgh handedness inventory) (Oldfield 1971); age: 18–50
years; minimum education level: 6 years of education;
remission lasting more than 6 months; CDAI score ≤ 150;
CDEIS score < 3.
Brain Imaging and Behavior (2018) 12:1795–1803
Exclusion criteria: abnormal levels of inflammation
(C reactive protein > 10 mg/L; erythrocyte sedimentation
rate > 20 mm/h; platelets > 300 × 10 9/L); history of CD-
related abdominal surgery; history of treatment with gluco-
corticoids, anti TNF-α drugs, psychotropic or opioid drugs
in the past 3 months; pregnant or breastfeeding women;
patients with current or previous history of neurological or
psychiatric conditions, head trauma or loss of conscious-
ness; claustrophobia; and patients with metallic implants in
the body.
Forty gender, age, and education matched healthy sub-
jects (HCs) were recruited from the Shanghai University of
Traditional Chinese Medicine via a newspaper advertise-
ment. They did not suffer from any gastrointestinal disorders
or pain and did not receive any medications. In addition,
they had negative results on colonoscopy performed within
the preceding year as part of routine physical examination.
All subjects were evaluated by an experienced gastroen-
terologist. To rule out psychiatric or neurological disorders,
psychiatric examinations were performed by an experienced
psychiatrist, based on a structured psychiatric interview tool
from the Diagnostic and Statistical Manual of Mental Dis-
orders, 4th edition (DSM-IV).
Symptom assessment
The condition of CD patients was assessed by the Crohn’s
disease activity index (CDAI) (Best et al. 1979); the quality
of life was assessed using the Inflammatory Bowel Disease
Questionnaire (IBDQ) (Irvine et al. 1994); emotional dis-
tress was assessed using the Hospital Anxiety and Depres-
sion Scale (HADS) (Zigmond and Snaith 1983).
MRI data acquisition
All MRI data were obtained from a 3T magnetic resonance
scanner (Siemens, TRIO, Erlangen, Germany) in the Depart-
ment of Radiology at the Shanghai Mental Health Center.
All subjects were supine on the scanner and were told to
relax, keep their eyes closed and not fall asleep or think. A
standard sponge pad was used to fill the gap between the
head and the coil to prevent head movement and to protect
the ears from the noise of scanner.
A set of high-resolution 3D T1-weighted structural
images was obtained prior to functional san (TR/TE:
2300 ms/2.98 ms; field of view (FOV): 256 × 256 m ­ m 2;
matrix size: 256 × 256; flip angle: 9°; in-plane resolution:
1 × 1 ­mm2; slice thickness: 1.0 mm with no gaps and 176
slices). Functional images were acquired with a single-shot
gradient–recalled echo planar imaging (EPI) sequence R/
TE: 2000 ms/30 ms; 180 time points; FOV: 240 × 240 ­mm2;
matrix size: 64 × 64; flip angle: 90°; in-plane resolution:
1797
3.75 × 3.75 ­mm2, 32 sagittal slices and slice thickness: 5 mm
with no gaps.
Image data preprocessing
The imaging data processing assistant for resting-state fMRI
(DPARSF, V3.1, http://www.restf​mri.net) was used to ana-
lyze imaging data (Chao-Gan and Yu-Feng 2010), which is
based on SPM 12 (http://www.fil.ion.ucl.ac.uk/spm/softw​
are/spm12​), and the resting state fMRI data analysis toolkit
(REST, V1.8, http://restf​mri.net/forum​/ REST_V1.8) (Song
et al. 2011). The first ten volumes of each functional time
series were discarded to allow for signals equilibrium and for
participants’ adaptation to the scanning noise. The remain-
ing images were corrected for temporal difference in the
acquisition of the different slices and then realigned to the
first volume for head-motion correction. Subjects with head
motion exceeded 2 mm translation or 2 degrees rotation in
any direction were excluded. The corrected images were
further spatially normalized to the Montreal Neurological
Institute (MNI) 152 template and resliced with isotropic
3 mm × 3 mm × 3 mm voxel size and spatially smoothed
with a 6-mm full-width at half maximum (FWHM) Gauss-
ian kernel. After the linear trend of time courses removal,
the imaging data were then temporally band pass filtered
(0.01–0.1 Hz) to remove the effects of low-frequency drift
and high-frequency noise. The Friston 24 head-motion
parameters, white matter signal, and cerebral spinal fluid
signal were regressed out as nuisance covariates (Fair et al.
2008).
Amplitude of low‑frequency fluctuation (ALFF)
ALFF maps were calculated using REST v1.8 software
(http://restf​mri.net/forum​/REST_V1.8) (Song et al. 2011).
First, the preprocessed time series was transformed to fre-
quency domain using fast Fourier transform (FFT) to obtain
power spectrum. Then, the square root was calculated at
each frequency of the power spectrum and averaged across
0.01–0.1 Hz at each voxel. This averaged square root was
taken as the ALFF. To reduce the global effects of variabil-
ity across the subjects, the ALFF of each voxel was divided
by the global mean ALFF value to standardize data across
subjects (Zang et al. 2007).
Functional connectivity (FC)
CD is an autoimmune disease and hippocampus is thought
to play an important role in neuroimmune regulation (Lathe
2001). Previous studies (Agostini et al. 2013; Bao et al.
2015, 2016a , b) have shown differences in gray matter
structures and functional activity in the hippocampal cortex
of patients with CD. This study also found abnormal ALFF
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values in bilateral hippocampal cortex. Thus, the detected
bilateral hippocampus clusters from the ALFF analysis were
used as the regions of interest (ROIs). The MNI coordinates
of the center of the 6-mm spherical ROI were determined by
the peak t-score detected at the hippocampus. For functional
connectivity analysis, the mean time series was extracted
from the seed region and correlated with the time series
of each voxel of the whole brain for each subject. The cor-
relation coefficients were transformed into z values using
Fisher’s r-to-z transformation to improve normality. An
entire brain z-score map was created for each subject (Wei
et al. 2016). In this study, the global mean timecourse was
not regressed out in the model.
Statistical analyses
Clinical and demographic characteristic measures were ana-
lyzed with Statistical Product and Service Solutions (SPSS)
16.0 software [SPSS Inc. Chicago, IL]. The data are pre-
sented as mean ± standard deviation.
In this study, we only focused on the group-difference-
related positive intrinsic connectivity, which was used to
identify abnormal FC connectivity within this specific brain
network. A two sample t-test was performed to assess the
differences of ALFF and FC between the two groups within
the brain network, with gender, age, anxiety score of HADS
(HADS-A) and the depression score of HADS (HADS-D)
as covariates. The inclusion of anxiety and depression as
covariates was to exclude the effects of negative emotions
on the results and to focus on the impact of the disease itself
on brain activity. The statistical significance was set at voxel-
wise P < 0.001 uncorrected with an extent threshold of clus-
ter-wise false discovery rate (FDR) (P < 0.05 at cluster lever,
cluster size > 45). ROI-wise analysis was used to investigate
the relationship between ALFF alteration and disease dura-
tion. Pearson’s correlation was calculated in each group at
a threshold of P < 0.05 with the Bonferroni correction. The
ROIs included all nine brain regions showing different ALFF
values in CD patients. The MNI coordinates of the center of
the 6-mm spherical ROI were determined by the peak t-score
of all detected ROIs.
Results
Clinical and demographic characteristics
Clinical and demographic characteristics of study subjects
are summarized in Table 1. CD patients and HCs did not dif-
fer significantly with respect to demographic characteristics
including gender, age, height, and weight (P > 0.05). The
CD patients had significantly higher HADS-A and HADS-D
scores than the HCs (both P < 0.01).
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Brain Imaging and Behavior (2018) 12:1795–1803
None of the subjects were smokers. 51.7% of the patients
presented with abdominal pain. Of these, 28 were treated
with mesalazine, 11 were treated with azathioprine, four
were treated with mesalazine plus azathioprine, and 17 did
not receive any medication.
Difference in ALFF values between the CD patients
and HCs
As showed in Table 2, compared to the HCs, the CD patients
showed higher ALFF values in bilateral hippocampus and
parahippocampus, left anterior cingulate cortex (ACC)
and precuneus, and right insula and superior frontal cortex
(Fig. 1a). The CD patients also showed lower ALFF val-
ues in left precentral gyrus, left medial prefrontal cortex
(MPFC), and right secondary somatosensory cortex (S2)
(Fig. 1b).
There was no significant correlation between the abnor-
mal ALFF values and disease duration in the patients with
CD (P > 0.05 for all).
Difference in functional connectivity
of hippocampus between the CD patients and HCs
As showed in Table 3, compared to HCs, the left hippocam-
pus of the CD patients had lower functional connectivity
with the left inferior temporal cortex, and right MCC, hip-
pocampus and fusiform (Fig. 2a). The right hippocampus
of the CD patients had lower functional connectivity with
the right inferior orbitofrontal cortex and left hippocampus
(Fig. 2b).
Discussion
The results showed significant differences in ALFF values
in multiple brain regions in CD patients, including higher
ALFF values in hippocampus and parahippocampus, ACC,
precuneus, insula, and superior frontal cortex, and lower
ALFF values in precentral gyrus, MPFC, and S2. Functional
connectivity in hippocampus-limbic system in CD patients
was also significantly lower than that in HCs. Together,
abnormal activities and connectivity in these brain regions
may suggest abnormal brain functions related to the regu-
lation of visceral sensation, pain processing, default mode
network (DMN), and neuro-immunity in these patients.
ALFF differences between patients with CD and HCs
In this study, CD patients showed different spontaneous fluc-
tuations in brain regions including insula, ACC, MPFC, pre-
central gyrus, S2 and hippocampal cortex, all of which are
important components of visceral sensory and pain networks
Brain Imaging and Behavior (2018) 12:1795–1803 1799

Table 1  Demographic and CD Patients Healthy controls P value

clinical characteristics of the (n = 60) (n = 40)
study population
Sex (male/female) 43/17 27/13 0.656
Age (years) 30.80 ± 7.62 30.68 ± 5.78 0.930
Height (cm) 169.80 ± 6.94 169.68 ± 7.03 0.930
Weight (kg) 56.48 ± 9.04 59.33 ± 6.07 0.85
HADS-A 5.85 ± 3.46 3.25 ± 1.98 0.000
HADS-D 4.57 ± 3.68 2.95 ± 1.83 0.012
Disease duration (months) 77.70 ± 50.40 — —
CDAI 76.61 ± 43.93 — —
IBDQ 173.53 ± 26.29 — —
PLT 220.93 ± 43.21 — —
ESR 10.93 ± 5.58 — —
CRP 4.48 ± 2.84 — —
CDEIS 1.14 ± 0.73 — —
Montreal classification
Age at diagnosis A1 6 — —
A2 53 — —
A3 1 — —
Location L1 14 — —
L2 12 — —
L3 34 — —
L4 0 — —
Behavior B1 20 — —
B2 4 — —
B3 17 — —
B1P 5 — —
B2P 1 — —
B3P 13 — —

Data presented as mean (± standard deviation)

CD Crohn’s disease, CDAI CD Activity Index, CDEIS CD Endoscopic Index of Severity, CRP C-reactive
protein, ESR eosinophil sedimentation rate, HADS The Hospital Anxiety and Depression Scale, HADS-A
The Anxiety score of HADS, HADS-D The Depression score of HADS, IBDQ In-flammatory Bowel Dis-
ease Questionnaire, PLT platelet levels

(Mayer et al. 2006; Van et al. 2007), and are involved in
visceral sensory (pain) and movement regulation. Precuneus
and MPFC are important elements of DMN (Buckner et al.
2008; Mantini and Vanduffel 2013). Our previous findings
regarding brain areas involved in altered regional homogene-
ity (ReHo) in CD patients during resting-state is consistent
with these results (Bao et al. 2016a, b).
Although the inflammation is relatively stable in patients
with CD in remission, long-term chronic inflammation may
have an impact on functional activities of brain owing to the
relapsing-remitting disease course. In CD patients, intesti-
nal inflammation / pain signals are transmitted through the
brain–gut axis to the brain, and may lead to alterations in
multiple functional brain networks (Bonaz and Bernstein
2013). Differences in spontaneous fluctuations in insula, cin-
gulate cortex, hippocampus, PFC and other brain areas asso-
ciated with visceral activity and pain regulation may suggest
different neural network activities involved in processing
visceral sensory and pain in these patients. Indeed, more
than a half of all CD patients present with abdominal pain/
abdominal distension due to long-term chronic inflammation
of the intestines, despite being in remission. In our previ-
ous study, we found different brain activities between CD
patients with and without abdominal pain (Bao et al. 2016),
which suggests that chronic abdominal pain in patients in
remission may affect functional activities of brain.
In addition, the spontaneous fluctuations in DMN were
also different in CD patients. Precuneus and MPFC are
all component of DMN. Spontaneous fluctuations were
lower in anterior DMN (MPFC), and higher in posterior
DMN (precuneus), which suggests lower coupling between
the anterior and posterior DMN in patients with CD. The
anterior DMN is mainly involved in monitoring one’s
own state, predicting nociceptive stimulus, and emotional

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Table 2  Brain regions with Regions Hem BA MNI t value Voxels

significant differences in ALFF
values between CD patients and X Y Z
HCs
CD > HC
Insula R 47 36 18 –9 4.76 60
ACC​ L 24 –6 21 24 3.73 111
HIPP/paraHIPP L 20 –36 –24 –6 3.92 50
HIPP/paraHIPP R 37 24 –30 –6 3.87 53
Precuneus L 19 –15 –69 39 3.96 87
Superior frontal cortex R 8 18 18 54 4.63 91
CD < HC
Precentral gyrus L 6 –48 –3 39 –3.83 52
MPFC L 10 –3 54 6 –3.89 132
S2 (Rolandic-Oper) R 48 48 –9 18 –3.99 63

The results employed age, gender, anxiety and depression as covariates. The statistical threshold was set at
P < 0.05 (false discovery rate corrected) at cluster level and the cluster size > 45
ACC​ anterior cingulate cortex, BA Brodmann area, CD Crohn’s disease, Hem hemisphere, MPFC medial
prefrontal cortex, HCs healthy controls, HIPP / paraHIPP hippocampal / parahippocampal cortex

Fig. 1  Brain regions exhibited

significant differences in ampli-
tude of low frequency fluctua-
tions (ALFF) values between
CD patients and healthy control
subjects using age, gender,
anxiety and depression as
covariates. The statistical sig-
nificance was set at voxel-wise
P < 0.001 uncorrected with an
extent threshold of cluster-wise
false discovery rate (FDR)
(P < 0.05 at cluster lever, cluster
size > 45). (a) Brain regions
exhibit significantly higher
ALFF values in CD patients;
(b) Brain regions exhibit
significantly lower ALFF values
in CD patients. ACC, anterior
cingulate cortex; CD, Crohn’s
disease; MPFC, medial prefron-
tal cortex; HC, healthy control;
HIPP / paraHIPP, hippocampal
/ parahippocampal cortex;
PCUN, precuneus; PREC,
precentral gyrus; S2, secondary
somatosensory cortex; SFC,
superior frontal cortex

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Brain Imaging and Behavior (2018) 12:1795–1803 1801

Table 3  Brain regions showing Regions Hem BA MNI T Value Voxels

significantly lower functional
connectivity to bilateral X Y Z
hippocampus between CD
patients and HCs Left hippocampus
MCC R 23 9 –21 36 -3.83 67
Hippocampus R 36 30 –3 –30 –3.75 49
Fusiform R 20 30 –3 –39 –3.87 147
Inferior temporal cortex L 20 –42 3 –36 –4.29 104
Right hippocampus
OFC R 47 39 42 –18 –4.53 87
Hippocampus L 20 –33 –24 –15 –3.67 52

The results employed age, gender, anxiety and depression as covariates. The statistical threshold was set at
P < 0.05 (false discovery rate corrected) at cluster level and cluster size > 45
BA Brodmann area, CD Crohn’s disease, Hem hemisphere, HCs healthy controls, MCC middle cingulate
cortex, OFC orbitofrontal cortex

Fig. 2  Brain regions showed significant differences in the hippocam-
pus-related connectivity between CD patients and healthy control
subjects using age, gender, anxiety and depression as covariates. The
statistical significance was set at voxel-wise P < 0.001 uncorrected
with an extent threshold of cluster-wise false discovery rate (FDR)
(P < 0.05 at cluster lever, cluster size > 45). (a) Brain regions show-
ing significantly lower connectivity to the left hippocampus in CD
patients; (b) Brain regions showing significantly lower connectivity
to the right hippocampus in CD patients. CD, Crohn’s disease; HC,
healthy control; HIPP, hippocampus; ITC, inferior temporal cortex,
MCC, middle cingulate cortex; OFC, orbitofrontal cortex

assessment or integration (Blakemore 2008; Wiech et al.
2005), while the posterior DMN is mainly associated
with self-introspection and episodic memory processing
(Cavanna and Trimble 2006; Margulies et al. 2009). These
results suggested that relevant DMN functions, monitoring
of the external environment and spontaneous cognition
processing (Buckner et al. 2008; Mantini and Vanduffel
2013), were different in CD patients.
Previously, we found lower gray matter volume in
MPFC, but higher gray matter volume in precuneus in
patients with CD. We speculated that the different gray
matter volume in CD patients may be the structural basis
of abnormal spontaneous activities.

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Differences in hippocampus‑related functional
connectivity between CD patients and HCs
Our study demonstrated different spontaneous functional
activities in hippocampal cortex, which is consistent with
findings of previous neuroimaging studies which showed
abnormal gray matter structure and function of hippocampus
in these patients (Agostini et al. 2013; Bao et al. 2015, 2016a,
b). The hippocampus plays an important role in neuroimmuno-
logical regulation by affecting cellular and humoral immunity
through the hypothalamic–pituitary–adrenal (HPA) axis and
neurohumoral pathways (Lathe 2001). Further, the hippocam-
pus can also interact with the vagus nerve to regulate the func-
tion of immune cells in the intestinal wall through the release
of acetylcholine, and, thereby, be involved in neural regula-
tion of intestinal inflammation. Studies in chemically induced
models of CD intestinal inflammation have shown increased
CNS excitability and behavioral changes, which may be related
to hippocampal microglia activation, increased expressions of
TNF-α, nitric oxide synthase (iNOS), and nitrite content (Hey-
darpour et al. 2016; Riazi et al. 2008), enhanced glutamatergic
synaptic transmission and plasticity, and reduced neurogenesis
(Riazi et al. 2015; Zonis et al. 2015). Therefore, we speculate
that intestinal immune dysfunction / inflammation may affect
the structure and function of hippocampal cortex in patients
with CD.
Whole-brain seed-based FC analysis using bilateral hip-
pocampus as seed regions showed lower functional connectiv-
ity between hippocampus and brain structures of the limbic
system. These brain regions are may be mainly involved in the
regulation of visceral functions (including visceral sensation
and pain). The result is consistent with the previous findings.
The limbic system is considered to be a higher center for
regulation of visceral functions. In particular, visceral func-
tional activity is regulated by hippocampus and other brain
regions of the limbic system (Drevets et al. 2008; Jones et al.
2006). Previous results have confirmed different spontane-
ous activities in networks related to visceral sensory and
pain in CD patients. As a key brain region involved in neu-
roimmune regulation, the hippocampus, together with other
brain structures of the limbic systems, regulates visceral
sensation and movement (Drevets et al. 2008; Jones et al.
2006). The finding of lower functional connectivity between
hippocampus and other brain structures of the limbic system
may suggest reduced ability of the limbic system in regulat-
ing visceral sensation and pain network.
Limitations and future directions
This study has several limitations. First, this is a cross-
sectional study, which makes it impossible to determine a
potential causal relationship between abnormal brain activity
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Brain Imaging and Behavior (2018) 12:1795–1803
and the occurrence of disease. Second, immunosuppressive
therapy may be a confounding factor that could not be con-
trolled in this study. This is probably a common limitation
for many fMRI studies (Mikdashi 2016).
In conclusion, this study found differences in spontaneous
fluctuations of activities of multiple brain regions in patients
with CD in remission. Since the combined functions of these
brain regions involve visceral sensation, pain processing, and
DMN, the observed different brain activity in CD patients
may reflect their abnormal functions in visceral sensation
and pain, external environment monitoring, and cognition
processing. In addition, functional connectivity between hip-
pocampus and visceral sensory and pain network was also
lower. These results may provide a better understanding of
the pathophysiological mechanisms of CD.
Acknowledgements Many thanks to Dr. Lili Ma at Zhongshan Hos-
pital of Fudan University for endoscopy examination and scoring.
This work was funded by the National Key Basic Research Program
of China (973 program), No. 2009CB522900, 2015CB554501; the
Program for Outstanding Medical Academic Leader, No. 80; the Pro-
gram of Shanghai Academic Research Leader, No. 17XD1403400, the
National Natural Science Foundation of China, No. 81471738 and the
National Institutes of Health grant (P20GM103472).
Compliance with ethical standards
Conflict of interest The authors disclose no conflicts.
Research involving human participants All procedures performed in
this study were in accordance with the ethical standards of the institu-
tional and/or national research committee and with the 1964 Helsinki
declaration and its later amendments or comparable ethical standards.
Informed consent Informed consent was obtained from every partici-
pant included in the study.
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